Method for the manufacture of bio-products with a modified sugar profile

ABSTRACT

The invention relates to a method for the production of a solid bio-product wherein at least 80% of the original indigestible oligosaccharide (raffinose, stachyose and verbascose) content has been degraded into digestible mono- and disaccharides, comprising the following steps: 1) providing a mixture of milled or flaked or otherwise disintegrated biomass, comprising oligosaccharides and optionally polysaccharides and further comprising proteinaceous plant parts, water and one or more enzyme preparations containing α-galactosidase(s); 2) reacting the mixture resulting from step (1) under continuous mixing and under conditions where the water content in the initial mixture does not exceed 65% by weight, for 0.15-36 hours at a temperature of about 20-65° C.; 3) incubating the reacted mixture from step (2) at a temperature and in a time period which inactivate said α-galactosidase(s), as well as solid bio-products obtainable by such method. The invention further relates to uses of the bio-product and a food, a feed, a cosmetic or pharmaceutical product or a nutritional supplement containing the solid bio-product.

FIELD OF THE INVENTION

The present invention relates to a method for the production ofbio-products with a modified sugar profile that is enriched inmonosaccharide and sucrose content and reduced in indigestibleoligosaccharide content. Further the present invention relates to amethod, where the fermentable sugars originally present or provided byα-galactosidase, may be further converted with a fungus or bacteria.

Furthermore it relates to the products obtainable by the method as wellas the use of the products obtained.

BACKGROUND OF THE INVENTION

There is a need for bio-products that primarily can be used asingredients in food or feed. The basic constituents in such products areproteins, fats and carbohydrates.

Suitable biomasses for such products are grasses and oil bearing crops,such as seeds, cereals and pulses. Cereals have a protein content up to15% e.g. wheat, and pulses have a content of up to 45% e.g. soybeans,based on dry matter.

A general problem especially related to pulses is the content ofindigestible oligosaccharides causing flatulence when fermented in thegut. The presence of the oligosaccharides raffinose, stachyose andverbascose can be reduced by soaking in water or enzymatically byhydrolysis with α-galactosidase. The problem associated with this isthat it adds to the cost of the final product due to the use of water insoaking or by an enzymatic treatment that has to be performed at arelatively high water content of 80% or more.

U.S. Pat. No. 6,238,725 B1 discloses a method for preparing a legumewhere the flatulence-causing oligosaccharides are removed by soaking inwater.

WO 02/15712 A2 discloses a method for manufacturing a soy proteinproduct by the use of α-galactosidase where the water content in theprocess is 80-90%. The soy product obtained has a protein content ofmin. 60% and a total content of raffinose and stachyose of less than 5%.

US 2003/019041 A1 discloses a method for the manufacture of a soyprotein concentrate by the use of a glycosidase enzyme (α-galactosidase)where the water content in the process according to the examples isapprox. 90%. After hydrolysis carbohydrates and salts are removed byultrafiltration. The soy concentrate obtained has a protein content ofmin. 65% and a combined content of raffinose and stachyose of less than4%.

WO 2009/143591 discloses a method for processing soybeans by the use ofenzyme(s) which is capable of converting the insoluble polysaccharidesinto soluble sugars and hydrolyzing the proteins at a water content ofmaximally 35%. The level of degradation is not disclosed.

The object of the present invention is to provide an improved method forthe production of bio-products with a modified sugar profile that isenriched in monosaccharide and sucrose content and reduced inindigestible oligosaccharide content, which can be performed at lowercosts due to the low water content in the process.

Yet another object is to provide a method where the fermentable sugarsoriginally present or provided by α-galactosidase, may be furtherconverted with a fungus e.g. yeasts and/or bacteria e.g. Lactobacillus.

These objects are fulfilled with the process and the products of thepresent invention.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a method for theproduction of a solid bio-product wherein at least 80% of the originalplant indigestible oligosaccharide (raffinose, stachyose and verbascose)content has been degraded into digestible mono- and disaccharidescomprising the following steps:

-   -   1) providing a mixture of milled or flaked or otherwise        disintegrated biomass, comprising oligosaccharides and        optionally polysaccharides, and further comprising proteinaceous        plant parts, water and one or more enzyme preparations        containing α-galactosidase(s);    -   2) reacting the mixture resulting from step (1) under continuous        mixing and under conditions where the water content in the        initial mixture does not exceed 65% by weight, for 0.15-36 hours        at a temperature of about 20-65° C.;    -   3) incubating the reacted mixture from step (2) at a temperature        and in a time period which inactivate said α-galactosidase(s).

It is surprising that the enzymatic hydrolysis i.e. catalyticdecomposition by reaction with water can be performed at a water contentthat does not exceed 65% by weight. Normally the low content of watertends to slow down the reaction due to mechanical contact problems withthe substrate. To give a scientific explanation at this stage would bespeculative. The combination of pre-treatment of the substrate, theactivity of the α-galactosidase and the mixing procedure during thereaction is believed to be the key factors.

The benefit is that the product resulting from the method only containsminor amounts of water due to the low water content during the process,and accordingly, drying of the product can be performed at low costs dueto the minor amount of water to be removed.

The invention further provides a solid bio-product obtainable by aprocess according to the invention comprising plant proteins in anamount up to about 60% and optionally glycerides up to 25% by weight ofdry matter.

The invention also provides a solid bio-product obtainable by a processas defined in the claims 13-20.

Finally, the invention provides a food, feed, cosmetic or pharmaceuticalproduct or a nutritional supplement containing from 1 to 99% by weightof a solid bio-product according to the invention.

DEFINITIONS

In the context of the current invention, the following terms are meantto comprise the following, unless defined elsewhere in the description.

The terms “about”, “around”, “approximately”, or “˜” are meant toindicate e.g. the measuring uncertainty commonly experienced in the art,which can be in the order of magnitude of e.g. +/−1, 2, 5, 10, 20, oreven 50%.

The term “comprising” is to be interpreted as specifying the presence ofthe stated part(s), step(s), feature(s), composition(s), chemical(s), orcomponent(s), but does not exclude the presence of one or moreadditional parts, steps, features, compositions, chemicals orcomponents. E.g., a composition comprising a chemical compound may thuscomprise additional chemical compounds, etc.

The term indigestible is to be interpreted as not digestible by humansand monogastric/non-ruminant animals.

The term “at least 80% of the original indigestible oligosaccharidecontent has been degraded” is to be interpreted as specifying that thetotal content of indigestible oligosaccharides has been degraded by atleast 80% and also includes products wherein one type of oligosaccharidemay be degraded to a larger extent than another type of oligosaccharide,and even wherein one type of oligosaccharide may be degraded only in aminor extent, as long as the total content of theoriginal—starting—oligosaccharides has been reduced as specified by atleast 80%.

Biomass:

Comprises biological material produced by the photosynthesis and thatcan be used as raw material in industrial production.

In this context, biomass refers to plant matter in the form of grasses,cereals, seeds, nuts, beans and peas, etc., and mixtures thereof.

Furthermore a biomass comprising pulses is preferred due to the proteincontent and composition. They also contain carbohydrates comprisingalpha-galactosides. In general the principal alpha-galactoside isstachyose except in field pea where the principal oligosaccharide isverbascose.

Otherwise Disintegrated:

Means disintegrated by cooking and/or by maceration and/or acid oralkaline pressure-cooking, or ultrasonic treatment.

Carbohydrates:

Comprise mono-, di-, oligo- and polysaccharides.

C5 sugars (pentoses) are carbohydrates where the component monomersugars are composed of a ring with five carbon atoms e.g. arabinose.

C6 sugars (hexoses) are carbohydrates where the component monomer sugarsare composed of a ring with six carbon atoms e.g. galactose.

Oligosaccharides and Polysaccharides:

An oligosaccharide is a saccharide polymer containing a small number(including up to e.g. 8-10) of component monomer sugars, also known assimple sugars. Typical examples are the trisaccharide raffinose(D-galactose-α1,6-D-glucose-α1,β2-D-fructose), the tetrasaccharidestachyose (D-galactose-α1,6-D-galactose-α1,6-D-glucose-α1,β2-D-fructose)and the pentasaccharide verbascose(D-galactose-α1,6-D-galactose-α1,6-D-galactose-α1,6-D-glucose-α1,β2-D-fructose).

Polysaccharides are saccharide polymers containing a large number ofcomponent monomer sugars, also known as complex carbohydrates. If themonomer sugars are of the same type the polysaccharide is called ahomopolysaccharide, but when more than one type is present they arecalled heteropolysaccharides.

Examples include storage polysaccharides such as starch and structuralpolysaccharides such as cellulose and arabinoxylan.

Proteinaceous Materials:

Comprise organic compounds made of amino acids arranged in a linearchain and joined together by a bond called a peptide bond. At a chainlength of up to approximately 50 amino acids the compound is called apeptide; at higher molecular weight the organic compound is called apolypeptide or a protein.

Fats:

Comprise esters between fatty acids and glycerol. One molecule ofglycerol can be esterified to one, two and tree fatty acid moleculesresulting in a monoglyceride, a diglyceride or a triglyceriderespectively. Usually fats consist of mainly triglycerides and minoramounts of lecithins, sterols, etc. If the fat is liquid at roomtemperature it is normally called oil. With respect to oils, fats andrelated products in this context, reference is made to “Lipid Glossary2”, F. D. Gunstone, The Oily Press, 2004.

Glycerides:

Comprise mono-, di- and triglycerides.

Processing Aids:

1. Enzymes

Enzyme(s) is a very large class of protein substances that act ascatalysts. Commonly, they are divided in six classes, and the mainclasses falling within the scope of this invention can be transferasesthat transfer functional groups and the hydrolases that hydrolyzevarious bonds.

In this context glycoside hydrolase enzymes are important and especiallyα-galactosidase(s) that is an enzyme that hydrolyses the terminalalpha-galactose in alpha-galactosides, comprising galactoseoligosaccharides and galacto-mannans, liberating D-galactose residues.

The activity of α-galactosidase in a preparation is expressed in units/gof actual enzyme product and assayed by reactingp-nitrophenyl-α-D-galactopyranoside with water and enzyme for 10 min at25° C. to form D-galactose and p-nitrophenyl that is monitoredspectrophotometrically at 410 nm.

One α-galactosidase unit is the enzymatic activity that liberates onemicromole (10⁻⁶ mole) of p-nitrophenyl per minute.

Further examples of enzymes comprise: protease(s), peptidase(s),β-galactosidase(s), amylase(s), glucanase(s), pectinase(s),hemicellulase(s), phytase(s), lipase(s), phospholipase(s) andoxido-reductase(s).

2. Plant Components and Organic Processing Agents

Some of the functional properties that are important in this contextare: Antioxidant, anti-bacterial action, wetting properties andstimulation of enzymes.

The list of plant-based components is huge, but the most important arethe following: Rosemary, thyme, oregano, flavonoids, phenolic acids,saponins and α- and β-acids from hops e.g. from humulone and lupolone,for the modulation of soluble carbohydrates. Furthermore organic acidse.g. Sorbic-, propionic-, lactic-, citric- and ascorbic acid and theirsalts for the adjustment of the pH-value, preservation and chelatingproperties is part of this group of processing aids.

3. Inorganic Processing Agents

Comprise inorganic compositions that are able to preserve the fermentingmixture against bacterial attack during processing e.g. Sodiumbisulfite, etc.

Anticaking and flow improving agents in the final product e.g. Potassiumaluminum silicate, etc.

Processed Food Products:

Comprise dairy products, processed meat products, sweets, desserts, icecream desserts, canned products; freeze dried meals, dressings, soups,convenience food, bread, cakes, etc.

Processed Feed Products:

Comprise ready-to-use feed or feed ingredients for animals such aspiglets, calves, poultry, furred animals, sheep, cats, dogs, fish andcrustaceans, etc.

Pharmaceutical Products:

Comprise products, typically in the form of a tablet or in granulatedform, containing one or more biologically active ingredients intendedfor curing and/or alleviating the symptoms of a disease or a condition.Pharmaceutical products furthermore comprise pharmaceutically acceptableexcipients and/or carriers. The solid bio products herein disclosed arevery well suited for use as a pharmaceutically acceptable ingredient ina tablet or granulate.

Cosmetic Products:

Comprise products intended for personal hygiene as well as improvedappearance such as conditioners and bath preparations.

DETAILED DESCRIPTION OF THE INVENTION

The original plant indigestible oligosaccharides the content of which isdegraded by the process of the invention are primarily raffinose,stachyose, and verbascose.

The water content in the initial reaction mixture does not exceed 65% byweight, which implies that the dry matter content in the mixture is atleast 35%.

The reaction time is 0.15-36 hours at a temperature of about 20-65° C.The temperature may e.g. vary from 25-60° C., 30-55° C., from 35 to 50°C., from 40 to 45° C.; the reaction time may e.g. vary from 10 minutesto 36 hours, from 20 minutes to 30 hours, from 1 to 24 hours, from 2 to20 hours, from 4 to 18 hours, from 8 to 16 hours or from 12 to 14 hours.

In one embodiment of the method of the invention it further comprisesthat fungus, such as live yeast, and/or bacteria is added to the biomasscomprising oligosaccharides and/or polysaccharides and proteinaceousplant parts in a dry matter ratio between fungus/bacteria and biomass offrom 1:2 to 1:400, and that the incubating in step (3) is carried out ata temperature and in a time period which inactivate saidα-galactosidase(s) and fungus and/or bacteria. Thus dry matter ratiossuch as: 1:2; 1:3; 1:4; 1:5; 1:6; 1:7; 1:8; 1:9; 1:10; 1:20; 1:30; 1:40;1:50; 1:60; 1:70; 1:80; 1:90; 1:100, 1:200 and 1:300 are included. Theincubation of the reacted mixture may be carried out at about 70-150°C., e.g. 85-150° C., including 70, 75, 80, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135,140, 145, 150° C. for 0.5-240 minutes, e.g. 6-240 minutes, including0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 90, 120, 150, 180, 210, and 240 minutes.

When choosing conditions for the incubation the skilled person knowsthat when using very high temperatures, relatively short incubationtimes are needed.

In this embodiment the reacting step (2) may be performed underanaerobic and/or aerobic conditions.

In a second embodiment the amount of the α-galactosidase preparation(s)is from 0.001 to 1.0% by weight of the biomass in the initial mixturewhere the α-galactosidase preparation(s) is having an activity of 5,000α-galactosidase units/g enzyme product, and/or the reaction in step (2)is performed under conditions where the water content in the initialmixture is from 30 to 65% by weight, which implies that the dry mattercontent in the mixture is from 35 to 70 by weight %.

Thus, the water content may vary to be e.g. from 35 to 60%, from 40 to55% or from 45 to 50%. Hence, the dry matter content in the reactionmixture provided in step (1) may vary correspondingly to be e.g. from 40to 65%, from 45 to 60% or from 50 to 55%, e.g. be 45%, 50, %, 55%,57.5%, 60%, 62.5%, 65% or 67.5%.

The amount of the one or more α-galactosidase preparation(s) may varye.g. from 0.01 to 1.0%, from 0.025 to 0.75%, from 0.05 to 0.5%, from0.075 to 0.25% or from 0.1 to 0.125% of an α-galactosidasepreparation(s) having an activity of 5,000 α-galactosidase units/genzyme product. The activity of the α-galactosidase preparation(s) mayalso be lower or higher than 5,000 α-galactosidase units/g enzymeproduct, e.g. from 5 to 200,000, or from 100,000 to 150,000, 50 to50,000 or from 500 to 10,000 units/g enzyme product, as long as theamount of enzyme applied is adapted to the strength of the enzymeproduct. The higher the amount and activity of the α-galactosidasepreparation the lower reaction time is generally needed and vice versa.The skilled person within enzyme technology will be aware of this.

In a third embodiment the reaction in step (2) is performed in one ormore non-vertical, interconnected paddle worm or continuous wormconveyers with inlet means for the reaction mixture and additives andoutlet means for the product as well as control means for rotationspeed, temperature and pH. This embodiment may be a variant wherein thecontinuous worm conveyer can be an optionally modified type of a singlebladed or multi bladed screw or intersected screw conveyer designed totransport the reacting mixture and at the same time lifting the materialso that it is transported and agitated without compacting it. Thereaction step (2) may also be performed in a Vertical Screw Mixer, e.g.a Nauta Mixer.

In a forth embodiment of the method of the invention one or moreprocessing aids, such as hop products containing α- and β-acids fromhops, are added in any of steps (1), (2) and/or (3). The one or moreprocessing aids may also be an enzyme, a plant component and/or organicprocessing agent and/or inorganic processing agent as defined above inthe definition section of the present application.

In a fifth embodiment the fungus and/or bacteria that may be added tothe biomass is live yeast selected among Saccharomyces cerevisiaestrains, including spent brewer's yeast and spent distiller's yeast andspent yeast from wine production and baker's yeast, as well as Bacilluscereus strains and yeast strains fermenting C5 sugars. The live yeastmay e.g. be added in an amount of from 0.25 to 10%, such as 0.5%, 1%,1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5, 5%, 6%, 7%, 8% or 9.

The proteinaceous plant parts comprised in the biomass may in a 6^(th)embodiment be pulses, such as soy, pea, bean, lupine, and/or cereals,such as wheat, and/or grasses and further be as defined in thedefinition section of the present application. The biomass may in a7^(th) embodiment further comprise oils and fats, e.g. from seeds of oilbearing plants, e.g. rape seed and soy, and further as defined above inthe definition section of the present application.

In an 8^(th) embodiment the reaction mixture resulting from step (3) isdried to a water content of no more than 10% by weight.

In further embodiments the amount of said one or more α-galactosidasepreparation(s) is from 0.25 to 1.0% by weight of the dry matter of thebiomass in the initial mixture where the α-galactosidase preparation ishaving an activity of 5,000 α-galactosidase units pr. g of enzymeproduct, and said reacting in step (2) is performed for 4-36 hours at atemperature of 30-60° C.; or

the amount of said one or more α-galactosidase preparation(s) is from0.25 to 1.0% by weight of the dry matter of the biomass in the initialmixture where the α-galactosidase preparation is having an activity of5,000 α-galactosidase units pr. g of enzyme product, and said reactingin step (2) is performed for 4-36 hours at a temperature of 50-60° C.;orthe amount of said one or more α-galactosidase preparation(s) is from0.01 to 1.0% by weight of the dry matter of the biomass in the initialmixture where the α-galactosidase preparation is having an activity of5,000 α-galactosidase units pr. g of enzyme product, and said reactingin step (2) is performed for 8-36 hours at a temperature 50-60° C.; orthe amount of said one or more α-galactosidase preparation(s) is from0.05 to 1.0% by weight of the dry matter of the biomass in the initialmixture where the α-galactosidase preparation is having an activity of5,000 α-galactosidase units pr. g of enzyme product, said reacting instep (2) is performed under conditions where the water content in theinitial mixture is from 40-65% and at a temperature of 50-60° C.; orfungus and/or bacteria is live yeast added in an amount of 0.25% to 10%.

In a final embodiment the method is performed as a batch, fed-batch orcontinuous process.

The solid bio-product obtainable by the process defined in claims 1 to16 comprises proteins in an amount up to about 60% by weight; the amountmay be less than 60%, e.g. 10-59%, 40-59%, 45-58%, 48-55% or 50-53% byweight of dry matter. The amount of glycerides may e.g. be 0-20%, 2-20%,5-18% or 10-15% by weight of dry matter.

The solid bio-product defined in claim 22 may comprise up to about 75%protein, e.g. from 40-75%, from 45-70%, from 48 to 65%, from 50-60% orfrom 53-55% protein by weight. It may further comprise glycerides in anamount up to 25% by weight of dry matter, e.g. 0-20%, 2-20%, 5-18% or10-15% by weight of dry matter.

The solid bio-product obtainable by the process of claims 11-16 maycomprise up to 60% protein by weight or it may comprise more than 60%protein by weight, e.g. from 40-75%, from 45-70%, from 48 to 65%, from50-60% or from 53-55% protein by weight. They may further compriseglycerides in an amount up to 25% by weight of dry matter, e.g. 0-20%,2-20%, 5-18% or 10-15% by weight of dry matter.

The amount of protein can be especially high when the biomass has beenfermented with live yeast according to the process of claim 2.

The oligosaccharides raffinose, stachyose and verbascose are degraded bythe method of the invention to mono- and disaccharides, such asgalactose and sucrose. Sucrose is an interesting product resulting ofthe method, because a high content of sucrose in the resultingbio-product will contribute to a satiated feeling after consumption. Theamount of sucrose may be as high as 15-20% by weight of dry matter.

In one embodiment the solid bio-product of the invention comprises atotal amount of raffinose, stachyose and verbascose of less than 8% byweight; e.g. less than 6%, less than 5%, less than 4%, less than 3%,less than 2 or less than 1% by weight.

In another embodiment the solid bio-product of the invention comprises atotal amount of raffinose of less than 3.0%, e.g. less than 2%, lessthan 1.5%, less than 1% or less than 0.75% or less than 0.5% or lessthan 0.25%.

In yet another embodiment the solid bio-product of the inventioncomprises a total amount of stachyose of less than 1.0%, e.g. less than0.75% or less than 0.5% or less than 0.25%.

The invention also relates to the use of a solid bio-product accordingto the invention in a processed food product for human and/or animalconsumption; as an ingredient to be used in a food or feed product; oras an ingredient of a cosmetic or a pharmaceutical product or anutritional supplement.

Finally the invention relates to a food, feed, cosmetic orpharmaceutical product or a nutritional supplement containing from 1 to99% by weight of a solid bio-product according to the invention.

EXAMPLES Example 1 Enzymatic Hydrolysis in a Laboratory Scale Process ofa Biomass Comprising Oligosaccharides from Soy 1.1 Materials and Methods

The enzymatic hydrolysis of the oligosaccharides stachyose and raffinosewas performed on 10 g of a biomass of defatted soy and water containingthe α-galactosidase enzyme added in an amount to reach a certain drymatter (DM) content of the mixture, and a certain enzyme concentration.

The mixing was performed to ensure homogeneity of the mixture.

The enzyme used in varying concentrations based on the dry matter of soybean meal was a commercial product from Advanced Enzyme Technologies,Maharasthra, India, marketed under the trade name SEBSoy 5.0 L.

The activity of SEBSoy 5.0 L is standardised to 5,000 U/g.

The enzymatic hydrolysis was performed in small glass containers at 34°C. and 55° C. for 4 to 16 hours followed by heat treatment at 100° C. toinactivate the enzyme.

After the enzymatic hydrolysis was terminated the content of solublecarbohydrates was extracted by stirring a watery suspension slurry of10% DM for 30 min followed by centrifugation for 10 min at 3,000×g.

The mono- and oligosaccharides in the watery extracts of the biomass wasanalyzed by thin layer chromatography on TLC silica gel 60 plates(Merck). The different components were quantified by comparison tostandards of known concentration. (Carbohydrate analysis—A practicalapproach; IRL Press, Oxford. Ed. M. F. Chaplan & J. F. Kennedy, 1986).

1.2 Results 1.2-a. Dose Response at Different Temperatures

The results listed in the following table were obtained after a reactiontime of 4 hours, at 45% DM:

Temperature 34° C. Temperature 55° C. Dose of SEBSoy Stachyose RaffinoseStachyose Raffinose Reference 6.0% 2.0% 6.0% 2.0% 0.05% — — 4.0% 3.0%0.10% 2.5% 3.5% 1.5% 2.5% 0.25% 1.5% 3.0% <0.25%  0.75%  0.50% 0.25% 1.0% — —

From the results it can be seen that stachyose is reduced at lowerenzyme concentrations as stachyose produce one molecule D-galactose andone molecule raffinose in the first step in the hydrolysis.

The total reduction of the combined content of stachyose and raffinoseis almost a linear function of the enzyme concentration.

Furthermore it can be seen that an increase in temperature from 34° C.to 55° C. corresponds to the same effect at a dose increase by a factorof 2 to 3.

1.2-b. Effect as a Function of Reaction Time

The results listed in the following table were obtained with a dose of0.05% SEBSoy, and at a reaction temperature of 55° C., at 45% DM:

Reaction time Stacchyose + In hours Stachyose Raffinose Raffinose 43.50% 4.00% 7.50% 8 0.75% 2.00% 2.75% 16 <0.25% 0.75% <1.00%

From the results it can be seen that every increase of the reaction timeby a factor of 2 reduces the combined content of stachyose and raffinoseby a factor of almost 3.

1.2-c. Effect as a Function of Dry Matter Content in the ReactionMixture

The results listed in the following table were obtained with a dose of0.25% SEBSoy, and at a reaction time of 4 hours at a temperature of 55°C.:

Dry matter in % Stacchyose + by weight Stachyose Raffinose Raffinose 500.25% 1.00% 1.25% 55 0.50% 1.25% 1.75% 60 0.75% 1.50% 2.25% 65 1.50%1.75% 3.25%

From the results it can be seen that there is an increase in the totalcontent of stachyose and raffinose as a function of dry matter and inthe interval 50 to 60% the increase is equidistant.

Example 2 Enzymatic Hydrolysis in a Laboratory Scale Process of aBiomass Comprising Oligosaccharides from Peas 2.1 Materials and Methods

The enzymatic hydrolysis of the oligosaccharides stachyose, raffinoseand verbascose was performed on 10 g of a biomass of milled pea andwater containing the α-galactosidase enzyme added in an amount to reacha dry matter (DM) content of the mixture of 50%, and a certain enzymeconcentration.

The enzyme preparation and the method used was as described in Example 1under paragraph 1.1

2.2 Results 2.2 Dose Response at Different Temperatures

The results listed in the following tables were obtained after areaction time of 4 hours, at 50% DM:

Temperature 34° C. Total oligo Dose of SEBSoy Stachyose RaffinoseVerbascose saccharides Reference 4.5% 0.5% 3.0% 8.0% 0.05% 3.5% 2.0%2.5% 8.0% 0.10% 3.0% 3.0% 2.0% 8.0% 0.25% 2.0% 2.0% 1.5% 5.5% 0.50% 0.5%1.5% 0.5% 2.5%

From the results it can be seen that using a 4 hour reaction time at 34°C. require a dose of 0.25% to get a reduction of the total content ofoligosaccharides.

Temperature 55° C. Total oligo Dose of SEBSoy Stachyose RaffinoseVerbascose saccharides Reference 4.5% 0.5% 3.0% 8.0% 0.05% 2.0% 1.5%1.5% 5.0% 0.10% 0.5% 1.5% 0.5% 2.5% 0.25% <0.25%  0.25%  <0.25%  <0.75% 0.50% <0.25%  <0.25%  <0.25%  <0.75% 

From the results it can be seen that by using a 4 hour reaction time at55° C. a dose of only 0.05% is required to get a reduction of the totalcontent of oligosaccharides. This corresponds to an increase inenzymatic activity by a factor about five when the reaction temperatureis raised from 34° C. to 55°.

Example 3 Enzymatic Hydrolysis in a Batch Process of a BiomassComprising Oligosaccharides and Proteins from Soy 3.1 Materials andMethods

200 kg of flash desolventized soy flakes were fed to a closed singlebladed worm conveyer able to transport, lift and mix the material. Atthe same time 170 liter of water and 200 ml of SEBSoy 5.0 L enzyme (0.1%dose of a preparation having an activity of 5,000 α-galactosidase unitspr. g of enzyme product) was added to reach a dry matter content ofabout 50% by weight in the mixture.

The mixture was hydrolyzed for 16 hours at 34° C. and dried to a watercontent of 5.6%. Watery extracts of the biomass was analyzed forcarbohydrate content by the phenol-sulphuric acid method andoligosaccharides were quantified after separation by TLC (Carbohydrateanalysis—A practical approach; IRL Press, Oxford. Ed. M. F. Chaplan & J.F. Kennedy, 1986).

3.2 Results

The results are tabulated in the following:

Subject Analytical values Protein in DM 59.6% Soluble carbohydrate 10.3%Stachyose <0.25%  Raffinose <0.25% 

From the results it can be seen that a product of the invention has aprotein content of about 60% by weight of dry matter and a low contentof oligosaccharides.

Example 4 Enzymatic Hydrolysis and Fermentation by Different Yeasts in aBatch Process of a Biomass Comprising Oligosaccharides and Proteins fromSoy 4.1 Materials and Methods

200 kg of flash desolventised soy flakes were fed to a closed singlebladed worm conveyer able to transport, lift and mix the material. Atthe same time 170 liter of water and a slurry of spent brewer's yeast orbaker's yeast and 200 ml of SEBSoy 5.0 L enzyme (0.1% dose of apreparation having an activity of 5,000 α-galactosidase units pr. g ofenzyme product) where added to reach a dry matter content of about 50%by weight in the mixture.

The two mixtures were hydrolyzed for 16 hours at 34° C. and dried to adry matter content of 95±0.3%.

The product was analyzed as in the previous example.

4.2 Results

The results are tabulated in the following:

Analytical values Analytical values Process with 3.5% Process with 1.0%spent brewer's yeast baker's yeast Subject added Added Protein in DM62.2%  61.9% Soluble carbohydrate 8.2%  7.2% Stachyose <0.25 <0.25%Raffinose <0.25 <0.25%

From the results it can be seen that a product of the invention has aprotein content slightly higher than 60% by weight of dry matter whenthe process is performed under yeast fermentation. The resulting producthas a low content of oligosaccharides.

Example 5 Comparative Enzymatic Hydrolysis in a Laboratory Scale Processof a Biomass Comprising Oligosaccharides from Soy

In this example the effects on the reduction of oligosaccharides byα-galactosidase under the processing parameters (temperature and drymatter) of the present invention vs. those of WO 2009/143591 isillustrated.

In WO 2009/143591 the water content is claimed to be maximum 35% orbetter 30% or even better 25%. The optimum temperature for the enzymatichydrolysis is mentioned to be between 60 and 80° C.

5.1 Materials and Methods

The enzymatic hydrolysis of the oligosaccharides stachyose and raffinosewas performed on 10 g of a biomass of defatted soy and full fat soy beanmeal and water containing the α-galactosidase enzyme added in an amountto reach a certain dry matter (DM) content of the mixture, and a certainenzyme concentration.

The mixing was performed to ensure homogeneity of the mixture.

The enzymes used in varying concentrations based on the dry matter ofdefatted soy or of soy bean meal was commercial products available fromAdvanced Enzyme Technologies, Maharasthra, India, marketed under thetrade name SEBSoy, and α-galactosidase from Enzyme DevelopmentCorporation (EDC) New York, USA.

The activity of the enzyme preparations was standardised to 5,000 U/g.

The enzymatic hydrolysis was performed in small glass containers at 55°C. or 70° C. for 4 and 16 hours followed by heat treatment at 100° C. toinactivate the enzyme.

After the enzymatic hydrolysis was terminated the content of solublecarbohydrates was extracted by stirring a watery suspension slurry of10% DM for 30 min followed by centrifugation for 10 min at 3,000×g.

The mono- and oligosaccharide content in the watery extracts of thebiomass was analyzed by thin layer chromatography on TLC silica gel 60plates (Merck). The different components were quantified by comparisonto standards of known concentration (Carbohydrate analysis—A practicalapproach; IRL Press, Oxford. Ed. M. F. Chaplan & J. F. Kennedy, 1986).

5.2 Results A. Present Invention Parameters: DM 45%, Temperature 55° C.and Reaction Time 16 Hours

Oligosaccharide Stachyose + reduction Biomass Enzyme Stachyose RaffinoseRaffinose In % Defatted soy —  6.0% 3.0%  9.0% 0 Defatted soy 0.05%SEBSoy <0.25% 0.75%   <1.0% >89 Defatted soy 0.05% EDC <0.25% 0.5%<0.75% >92 Full fat soy —  5.0% 2.0%  7.0% 0 Full fat soy 0.05% SEBSoy<0.25% 0.5% <0.75% >89 Full fat soy 0.05% EDC <0.25% <0.25%   <0.5% >92

From the results it can be seen that at the processing parameters of thepresent invention the reduction of oligosaccharides in the biomass ishigher than 89% after a reaction time of 16 hours at an enzyme dose of0.05%.

B. Present Invention Parameters: DM 45%, Temperature 55° C. and ReactionTime 4 Hours

Oligosaccharide Stachyose + reduction Biomass Enzyme Stachyose RaffinoseRaffinose In % Defatted soy —  6.0%  3.0% 9.0% 0 Defatted soy 0.25%SEBSoy <0.25% <0.25% <0.5% >94 Defatted soy 0.25% EDC <0.25% <0.25%<0.5% >94 Full fat soy —  5.0%  2.0% 7.0% 0 Full fat soy 0.25% SEBSoy<0.25% <0.25% <0.5% >93 Full fat soy 0.25% EDC <0.25% <0.25% <0.5% >93

From the results it can be seen that at the processing parameters of thepresent invention the reduction of oligosaccharides in the biomass ishigher than 93% after a reaction time of 4 hours at an enzyme dose of0.25%.

C. WO 2009/143591 Parameters: DM 70%, Temperature 70° C. and ReactionTime 4 Hours

Oligosaccharide Stachyose + reduction Biomass Enzyme Stachyose RaffinoseRaffinose In % Defatted soy — 6.0% 3.0% 9.0% 0 Defatted soy 0.25% SEBSoy4.0% 3.0% 7.0% 22 Defatted soy 0.25% EDC 2.5% 3.0% 5.5% 39 Full fat soy— 5.0% 2.0% 7.0% 0 Full fat soy 0.25% SEBSoy 3.0% 2.5% 5.5% 21 Full fatsoy 0.25% EDC 3.0% 2.0% 5.0% 28

From the results it can be seen that at the processing parameters of WO2009/143591 the reduction of oligosaccharides in the biomass lies in theinterval 21-39% after a reaction time of 4 hours at an enzyme dose of0.25%.

D. WO 2009/143591 Parameters: DM 70%, Temperature 70° C. and ReactionTime 16 Hours

Oligosaccharide Stachyose + reduction Biomass Enzyme Stachyose RaffinoseRaffinose In % Defatted soy — 6.0% 3.0% 9.0% 0 Defatted soy 0.05% SEBSoy6.0% 3.0% 9.0% 0 Defatted soy 0.05% EDC 3.5% 3.0% 6.5% 28 Full fat soy —5.0% 2.0% 7.0% 0 Full fat soy 0.05% SEBSoy 5.0% 2.0% 7.0% 0 Full fat soy0.05% EDC 4.0% 2.0% 6.0% 14

From the results it can be seen that at the processing parameters of WO2009/143591 the reduction of oligosaccharides in the biomass lies in theinterval 0-28% after a reaction time of 16 hours at an enzyme dose of0.05%.

5.3 Conclusion

From the results it is clear that the processing parameters used in WO2009/143591 (temperature and dry matter content) are not able to resultin the transformation of 80% or more of the oligosaccharides present ina biomass originating from soy; thus it is not possible to attain thesame high level of degradation into digestible mono- and disaccharidesas can be obtained by the method of the invention.

The invention claimed is:
 1. A method for the production of a solidbio-product having a modified sugar profile, comprising: (1) reacting aninitial mixture of milled or flaked or otherwise disintegrated biomasscomprising oligosaccharides and optionally polysaccharides and furthercomprising proteinaceous plant parts, water, and one or more enzymepreparations containing α-galactosidase(s), for 0.15-36 hours at atemperature of about 20-65° C. under continuous mixing, wherein theinitial mixture comprises an amount of said one or more enzymepreparations to provide α-galactosidase(s) in an amount equivalent tofrom 0.01 to 1.0% by weight, based on the dry matter weight of thebiomass, of an α-galactosidase preparation having an activity of 5,000α-galactosidase units per gram, wherein the amount of theα-galactosidase preparation is effective to convert at least 80% of theoriginal indigestible oligosaccharide content of the biomass intodigestible mono- and disaccharides, and wherein the water content in theinitial mixture does not exceed 65% by weight of the initial mixture;and (2) incubating the reacted mixture at a temperature and for a timeperiod which inactivate said α-galactosidase(s), thereby obtaining abio-product having a modified sugar profile, wherein at least 80% of theoriginal indigestible oligosaccharide content of the biomass has beendegraded into digestible mono- and disaccharides.
 2. The methodaccording to claim 1, further comprising adding fungus and/or bacteriato said biomass in an amount that results in a dry matter ratio offungus/bacteria to said biomass of from 1:2 to 1:400, wherein saidincubating is carried out at a temperature and for a time period whichinactivate said α-galactosidase(s) and fungus and/or bacteria.
 3. Themethod according to claim 2, wherein said reacting is performed underanaerobic and/or aerobic conditions.
 4. The method according to claim 1,wherein the water content in the initial mixture is from 30 to 65% byweight.
 5. The method according to claim 1, wherein said reacting isperformed in one or more non-vertical, interconnected paddle worm orcontinuous worm conveyers with an inlet for the mixture and additives,an outlet for the product, and control(s) for rotation speed,temperature and pH, or in a Vertical Screw Mixer.
 6. The methodaccording to claim 5, wherein said continuous worm conveyer is anoptionally modified type of a single bladed or multi bladed screw orintersected screw conveyer designed to transport the reacting mixtureand at the same time lift the material so that it is transported andagitated without being compacted.
 7. A method according to claim 1,wherein the initial mixture further comprises one or more processingaids selected from hop products containing α- and β-acids from hops, orsaid one or more processing aids is/are added during said reacting. 8.The method according to claim 2, wherein said fungus and/or bacteria islive yeast selected from Saccharomyces cerevisiae strains.
 9. The methodaccording to claim 1, where said proteinaceous plant parts comprisepulses.
 10. The method according to claim 1, where said biomass furthercomprises oils and fats.
 11. The method according to claim 1, where theobtained bio-product is dried to a water content of no more than 10% byweight.
 12. The method according to claim 1, where the reacting occursat 20-55° C.
 13. The method according to claim 1, wherein the amount ofsaid α-galactosidase preparation(s) in the initial mixture is equivalentto from 0.25 to 1.0% by weight, based on the dry matter weight of thebiomass in the initial mixture, of an α-galactosidase preparation havingan activity of 5,000 α-galactosidase units per gram, and said reactingis performed for 4-36 hours at a temperature of 30-60° C.
 14. The methodaccording to claim 1, wherein the amount of said α-galactosidasepreparation(s) in the initial mixture is equivalent to from 0.25 to 1.0%by weight, based on the dry matter weight of the biomass in the initialmixture, of an α-galactosidase preparation having an activity of 5,000α-galactosidase units per gram, and said reacting is performed for 4-36hours at a temperature of 50-60° C.
 15. The method according to claim 1,wherein the amount of said α-galactosidase preparation(s) in the initialmixture is equivalent to from 0.01 to 1.0% by weight, based on the drymatter weight of the biomass in the initial mixture, of anα-galactosidase preparation having an activity of 5,000 α-galactosidaseunits per gram, and said reacting is performed for 8-36 hours at atemperature 50-60° C.
 16. The method according to claim 1, wherein theamount of said α-galactosidase preparation(s) in the initial mixture isequivalent to from 0.05 to 1.0% by weight, based on the dry matterweight of the biomass in the initial mixture, of an α-galactosidasepreparation having an activity of 5,000 α-galactosidase units per gram,the water content in the initial mixture is from 40-65%, and saidreacting is performed at a temperature of 50-60° C.
 17. The methodaccording to claim 2, wherein the fungus and/or bacteria is live yeastadded in an amount of 0.25% to 10% by weight of the biomass.
 18. Themethod according to claim 1, performed as a batch, fed-batch orcontinuous process.
 19. The method according to claim 8, wherein saidfungus and/or bacteria is live yeast selected from the group consistingof spent brewer's yeast, spent distiller's yeast, spent yeast from wineproduction, baker's yeast, and yeast strains fermenting C5 sugars. 20.The method according to claim 1, where said proteinaceous plant partscomprises proteinaceous plant parts from one or more selected from thegroup consisting of soy, bean, pea, lupine, cereals, seeds, and grasses,which plant parts optionally may be defatted.
 21. The method accordingto claim 10, where said oils and fats are from seeds of oil bearingplants.
 22. The method according to claim 21, where said oil bearingplants are selected from the group consisting of rapeseed and soy.